FOLATE AND ONE-CARBON METABOLISM Folate is essential for one-carbon (methyl group) metabolism, a biochemical pathway involved in DNA synthesis, repair, and methylation. Efficient one-carbon metabolism also requires vitamins B-6 and B-12, riboflavin, and optimal activity of 10-20 enzymes. In a community-based, case-control study of invasive cervical cancer, we showed that high serum homocysteine was associated with a statistically significant 100-200% increase in risk and low serum or red blood cell folate, with only a 20-60% increase. This pattern suggests that circulating homocysteine may be an integratory measure of insufficient folate in tissues or a biomarker of disruption of one-carbon metabolism. Variant forms of two common polymorphisms in the methylene tetrahydrofolate reductase gene and a common polymorphism in the methionine synthase gene were each associated with elevated cervical cancer risk. Risk generally increased as copies of the variant gene increased. These results suggest that both genetic variability in the one-carbon metabolism pathway and micronutrient inadequacy can contribute to increased risk of cervical cancer. Additional polymorphisms in pathway genes are now being assayed. We are also exploring the role of one-carbon metabolism in the etiology of both colorectal and breast cancer in the Prostate, Lung, Colorectal, and Ovarian Cancer (PLCO) cohort. The large number of advanced colorectal adenomas (1200) identified will allow us to systematically search for main effects of polymorphic variation in key one-carbon metabolism genes, using spaced polymorphisms as biomarkers of genetic change. The relationships among circulating levels of homocysteine and various folate forms and genotype will also be explored. VEGETABLES, FRUITS, AND CAROTENOIDS The protective effect of vegetables and fruits is frequently touted as the most persuasive finding to emerge from epidemiologic studies of diet and cancer, with evidence strongest for lung and colorectal cancer. Individual carotenoids, measured in diet or blood, are reliable measures of intake of a variety of vegetables and fruits. In a nested case-control study of lung cancer in a cohort of Hawaiian Japanese men, individual carotenoids were measured in prediagnostic sera. Low serum levels of beta-cryptoxanthin, lycopene, and alpha-carotene, but not beta-carotene, were each modestly associated with elevated lung cancer risk (smoking-adjusted RRs = 1.3-1.5). There was no evidence of combined or synergistic effects for individual carotenoids. Thus, carotenoids, at physiologic levels, may not contribute substantially to lung cancer prevention. In the PLCO screening trial, we are investigating the relationship of vegetable and fruit intake, with quantity and variety assessed in several ways, to risk of colorectal adenoma. Greater intake of fruits and some vegetables, particularly deep yellow and dark green vegetables, is modestly, but significantly, associated with decreased risk for colorectal adenoma. Pyramid servings, a more comprehensive and quantitative approach to estimating food group intake, did not produce substantially different results than the more traditional number of servings/day. BREAST CANCER AND PROSTATE CANCER IN ASIAN-AMERICAN POPULATIONS International variation in breast cancer incidence and migrant studies indicate that modifiable factors play a major role in breast cancer etiology although the specific lifestyles and environmental exposures remain elusive. We designed a large, population-based case-control study of breast cancer in Asian-American women to take advantage of their diversity in lifestyle and breast cancer risk. Childhood, adolescent, and adult exposures were assessed by interviewing both study participants and their mothers. We observed a six-fold gradient in breast cancer incidence by migration patterns, comparable to the international differences in breast cancer incidence rates. Using the blood and urine samples that were collected, we initially focused on relationships, in the controls, between endogenous hormones and migration patterns. Estrogens did not differ significantly between Asian-American women born in Asia and the West. However, androgen levels were higher among Asian-Americans born in Asia. Thus, further efforts to understand the hormonal mechanisms underlying breast carcinogenesis should consider androgens as well as estrogens. The ratio of 2-hydroxyestrone to 16alpha-hydroxyestrone, an indicator of estrogen metabolism pathways, was consistently lower (by 20%) in Asian-American women born in the West. The 2:16alpha ratio may reflect Asian lifestyles that influence estrogen metabolism and reduce breast cancer risk. We are now analyzing insulin-like growth factors (IGFs). Adiposity and weight gain in the decade preceding diagnosis, as well as height, were critical determinants of breast cancer risk in these Asian-American women. We are seeking biologic explanations, with emphasis initially on the IGFs. Childhood, adolescent, and adult soy intake were each independently associated with reduced risk of breast cancer. The strongest effect was seen for childhood soy intake, with a statistically significant 60% reduction in risk between extreme tertiles. Further analysis of the dietary and cultural pattern information collected will clarify whether soy seems uniquely protective or may only be serving as an indicator of other Asian lifestyles. Preliminary results suggest that the relative risk of breast cancer for a positive family history is similar in Asian-American women at different levels of Westernization. This constancy implies that the lifestyles responsible for lower risk among Asians may also modify genetically determined breast cancer risk. Foci of prostate cancer cells, like foci of breast cancer cells, seem to advance more rapidly in Western societies than in Asian societies. Elevated IGF-I has been postulated as a biomarker of more rapid progression, and possibly a cause. With prospectively stored serum samples from a cohort of Hawaiian Japanese men, we are exploring the relationship of IGF-I, IGFBP-3, free IGF-I, PSA, and proteomic patterns to risk of prostate cancer. Two important questions are whether IGF-I is equally predictive of indolent and aggressive prostate cancer and whether it is a true risk factor, preceding diagnosis by many years, or simply an early marker of preclinical disease. ENDOGENOUS HORMONE MEASUREMENT We have published a series of manuscripts on the reproducibility and utility of the commercial kits currently used to measure estrogens, estrogen metabolites, and androgens in blood and urine from premenopausal women, postmenopausal women, and men. While some of the assays are sufficiently reliable to discriminate among individuals, others are more problematic. In collaboration with Drs. Xia Xu and Timothy Veenstra at NCI-Frederick and Dr. Larry Keefer in the NCI Center for Cancer Research, we have developed a robust, relatively rapid liquid chromatography/mass spectroscopy procedure that can measure simultaneously 16 estrogens and estrogen metabolites in 0.5 ml of urine. A formal test of reproducibility, sensitivity, and specificity in a range of samples is being implemented. We anticipate that our approach can be extended to estrogen metabolite measurement in serum/plasma and tissue and that it can be modified to also measure androgens and phytoestrogens. NATIONAL HEALTH EPIDEMIOLOGIC FOLLOW-UP STUDY With prospective dietary data from the nationally representative U.S. Health Examination Epidemiological Follow-up Study, we used principal component analysis to explore the role of dietary patterns in the etiology of prostate cancer.